Method of testing for diabetes that reduces the effect of interfering substances

ABSTRACT

An improved method of testing individuals for diabetes, even if they have levels of interfering substances (e.g., uric acid, bilirubin, and glutathione) that would otherwise interfere with such testing, is disclosed. The individual&#39;s protein-bound glucose level and glucose level are compared to the analogous values for a reference population to enable the risk of that individual&#39;s having diabetes to be assessed. The substances that would otherwise tend to interfere with the assay for protein-bound glucose are removed before the assay, desirably by precipitating the protein-bound glucose using uranyl acetate, which desirably leaves substantially all of the interfering substances in the supernatant, then separating the precipitate from the supernatant, redissolving the precipitate, and conducting the colorimetric assay on the resulting solution. An improved colorimetric test for protein-bound glucose using viologens as the colorimetric electron acceptors is also disclosed.

RELATED APPLICATIONS

This application is a continuation-in-part of (a) U.S. application Ser.No. 07/651768, filed Feb. 7, 1991, and (b) U.S. application Ser. No.08/014060, filed Feb. 5, 1993, which is a continuation of U.S.application Ser. No. 07/795990, filed Nov. 22, 1991, which is acontinuation-in-part of U.S. application Ser. No. 07/651768, filed Feb.7, 1991, all by the same inventors and all of which applications arehereby incorporated by reference in their entireties for all purposesand all of which are now abandoned.

BACKGROUND OF THE INVENTION

This invention relates to an improved test for diabetes mellitus("diabetes") that can be used even with a patient having levels ofsubstances that would otherwise interfere with testing, e.g., elevated(high) uric acid/and or bilirubin levels. Broadly speaking, thisinvention concerns a method of testing that treats a specimen from anindividual to substantially reduce or eliminate interfering substancesfrom the specimen, assays a material derived from the treated specimenfor a certain clinical value (protein-bound glucose level), obtains asecond clinical variable for that individual (glucose level), and thenuses those two clinical values to assess the likelihood of thatindividual having diabetes, e.g., by placing the individual in any oneof several categories, which categories are associated withpre-established risks of having diabetes.

As is well known, diabetes is a serious disease affecting a significantportion of the population. Detecting whether an individual has diabetesand monitoring diabetes therapy are some of the problems confrontingmedicine. An early screening test for diabetes and one that is stillcommonly used involves determining an individual's blood glucose level.See, e.g., U.S. Pat. Nos. 2,981,606; 3,653,841; 3,791,988; and 3,920,580(all of the patent and other documents, including literature articles,cited or otherwise identified in this application are herebyincorporated herein in their entireties for all purposes). Glycosylatedamino acids in urine have also been used to screen for diabetes. SeeU.S. Pat. No. 4,371,374. U.S. Pat. No. 4,397,956 concerns asingle-reading pseudo-kinetic method for monitoring the status ofcontrol of ketoacidosis-prone diabetics by measuring the blood glucoseand at least one additional indicator analyte (e.g., ketone bodies suchas acetone, beta-hydroxybutyrate, and acetoacetate and fatty acidderivatives).

One problem with glucose tests is that even in an individual who doesnot have diabetes, his or her glucose level can vary over a wide range,depending on when the test sample or specimen (e.g., blood) was takenand when and what the individual last ate. Furthermore, even if oneglucose test gives a high enough reading to strongly suggest thepresence of diabetes, that individual must undergo additional tests, forexample, a so-called glucose tolerance test, before a diagnosis ofdiabetes can be confirmed. The glucose level of a diabetic takinginsulin can also vary dramatically depending upon when the individuallast took insulin and on the dosage. It is not unknown for individualswho have diabetes to take insulin shortly before specimens are takenfrom them for therapy-monitoring glucose tests, so that their glucosetests will indicate normal levels of glucose and make it appear that theindividuals have been conscientiously following their prescribedregimens of insulin therapy. That makes monitoring such therapy moredifficult. For all these reasons, glucose testing alone was and is knownto have significant disadvantages.

Reactions of sugars and amino compounds (for example, proteins) to formN-substituted glycosylamines, which undergo subsequent irreversibleAmadori rearrangement, have been known for decades. See, e.g., Hodge,Agricultural And Food Chemistry, vol. 1, no. 15, pp. 928-943 (Oct. 14,1953). Hodge also noted at p. 930 that investigators showed that glucoseand the free amino groups of bovine serum albumin and other proteins andpeptides combined in a one-to-one molar ratio.

Years ago it was discovered that in a human diabetic, hemoglobin reactswith glucose in the blood to produce glycated (glycosylated) hemoglobinand that the level of glycated hemoglobin can be determined and used todetect diabetes and to monitor the course of therapy. Hemoglobin is themost abundant protein found in whole blood and its half-life is about 60days. Glycated hemoglobin forms when glucose binds to the amino moietiesof the hemoglobin. The bound glucose moiety undergoes Amadorirearrangement to form a fructose moiety. Both glucose and fructose arereducing sugars, that is, they can reduce other compounds (donateelectrons to the other compounds) under the appropriate reactionconditions. Because of the relatively lengthy half-life of hemoglobin,glycated hemoglobin is insensitive to short-term variations in glucoselevels, such as might be caused by taking a large dose of insulin oreating sugar-containing candy. Thus, it was discovered that the glycatedhemoglobin level indicated an individual's long-term blood glucosehistory. See, e.g., U.S. Pat. Nos. 4,200,435; 4,243,534; 4,260,516;4,268,270; 4,269,605; 4,399,227; 4,407,961; and 4,409,335.

There are other proteins in blood besides hemoglobin, and years ago itwas discovered that in a diabetic those other proteins also becomeglycated to a greater or lesser degree. Those proteins are reported tohave half-lives of anywhere from 2.5 to 23 days. In particular, thehalf-life of albumin is reported to be 14 to 20 days and will be takenas 19 days for purposes of further discussion. Albumin (at about 35-50grams per liter) and globulins (at about 20-30 grams per liter) are theprincipal proteins in serum. (Removal of blood cells from whole bloodyields plasma, and coagulation of the fibrinogen in the plasma andremoval of the resulting fibrin yields serum.) When serum proteins areglycated in vivo, glycated albumin usually accounts for about 80 percentof the glycated serum proteins (that is, protein-bound glucose).

Accordingly, detection of serum glycated proteins (sometimes called"fructosamines" in the literature) became another suggested method fordetecting diabetes and monitoring its therapy. See, for example, U.S.Pat. Nos. 4,642,295; 4,645,742; 4,797,473; 4,956,301; 5,055,388; JapanPatent Application No. 63-180861; Schleicher et al., J. Clin. Chem.Clin. Biochem., vol. 19, pp. 81-87 (1981); Dolhofer et al., ClinicaChimica Acta, vol. 112, pp. 197-204 (1981); Johnson et al., ClinicaChimica Acta, vol. 127, pp. 87-95 (1982); Armbruster, Clin. Chem., vol.33, no. 12, pp. 2153-2163 (1987); Rosenthal et al., Clin. Chem., vol.34, no. 2, pp. 360-363 (1988); Caines et al., Clin. Biochem., vol. 19,pp. 26-30 (February. 1986); Jue et al., J. Biochem. Biosphys. Meth.,vol. 11, pp. 109-115 (1985); Armbruster, Clin. Lab. Sci., vol. 3, no. 3,pp. 184-188 (May/June 1990); and Windeler et al., J. Clin. Chem. Clin.Biochem., vol. 28, pp. 129-138 (1990).

Various methods have been used to detect fructosamine, which is referredto herein as "protein-bound glucose" or "PBG." Use of the word"fructosamine" to refer to a protein-glucose Amadori rearrangementproduct is undesirable in part because it is not a recognized AmericanChemical Society or Chemical Abstracts protein chemical name or categoryand because it is also the trivial chemical name of a relatively simplecompound unrelated to proteins or protein-sugar adducts. Hence the term"protein-bound glucose," which is more accurate and whose meaning isclear, is preferred and is used herein. Current procedures for measuringserum glycated proteins (i.e., protein-bound glucose) include affinitychromatography, agarose gel electrophoresis, high-performance liquidchromatography (HPLC), immunoassay with monoclonal or polyclonalantibodies, and colorimetric methods.

It was known that under the appropriate conditions, typically alkalinepH, certain compounds that were otherwise colorless in solution would bereduced by (that is, receive electrons from) the reducing sugar moietyof the protein-bound glucose and become colored. It was also known thatthe reaction conditions could be chosen so that the intensity of thecolor would be directly proportional to the concentration ofprotein-bound glucose in the sample being tested. For example,tetrazolium and other compounds have been used as color indicators. See,e.g., U.S. Pat. Nos. 3,576,815; 3,791,988; 4,642,295; 4,645,742;4,956,301; Mattson et al., Anal. Chem., vol. 22, no. 1, pp. 182-185(January. 1950); Mopper et al., Anal. Biochem., vol. 45, pp. 147-153(1972); Chem. Abstr., vol. 95, entry 148753e (Foods, 1981); Caines,Clin. Biochem., vol. 19, pp. 26-30 (February 1986); Jue et al., J.Biochem. Biophys. Meth., vol. 11, pp. 109-115 (1985); and Chem. Abstr.,vol. 82, entry 70120f (Biochem. Meth., 1975). An assay marketed by RocheDiagnostic Systems, Inc., Montclair, N.J., uses nitroblue tetrazolium("NBT"). An assay marketed by Isolab Inc., Akron, Ohio, uses2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyltetrazolium chloride ("INT").Trudinger, Anal. Biochem., vol. 36, pp. 222-224 (1970), reports thatmethyl viologen is reduced by alkaline glucose to form a coloredsolution. Other compounds are known (including benzyl viologen) thatchange color and color intensity under the appropriate reactionconditions in the presence of reducing sugars or moieties of reducingsugars attached to proteins.

Johnson et al., Clinica Chimica Acta, vol. 127, pp. 87-95 (1982), reportthe advantages of measuring protein-bound glucose as an index ofdiabetic control. However, one recent review article concludes, basedupon an analysis of literature articles concerning this type of test,that the test has not been evaluated sufficiently to allow its routineclinical use and that the results reported for it in the literature donot suggest that the test is reliable. See Windeler et al., J. Clin.Chem. Clin. Biochem., vol. 28, pp. 129-138 (1990). The manufacturer ofone commercial assay recently referred to the growing unease with thefructosamine assay, said that people are beginning to feel that themethod has not lived up to its promise, and noted three technicalproblems with it. Isolines, vol. 19, no. 3, p. 1 (Isolab Inc., October1990).

The literature also reports that various substances can interfere withassays (and particularly colorimetric assays) for protein-bound glucose,e.g., non-specific binding agents and, if elevated, uric acid andbilirubin. Interference is one of the three problems noted by Isolab(Id.). Elevated uric acid and/or bilirubin can be the result of diseaseor other process (e.g., liver disease, cancer). Approximately 1% of thegeneral population and 6-7% of the hospital population have elevatedlevels. Colorimetric tests for protein-bound glucose in individualshaving sufficiently elevated bilirubin and/or uric acid levels areviewed as unreliable and such tests are generally not run for thoseindividuals.

U.S. Pat. Nos. 4,642,295 and 4,645,742, which use the coloring agentnitroblue tetrazolium ("NBT") under alkaline conditions to indicate thepresence and concentration of protein-bound glucose ("PBG"), requirethat two colorimetric intensity readings be taken to try to reduce theadverse effects of certain interference. The first reading is takenafter a suitable delay from the addition of NBT to the specimen (e.g.,10 minutes) to allow non-PBG/tetrazolium reactions to occur and thesecond reading is taken after a suitable delay following the firstreading. If the first intensity reading is taken before substantiallyall of those non-specific (i.e., non-PBG) reactions have occurred, thedifference in intensities between the two readings will include theintensity change caused by reaction of non-PBG compounds with thetetrazolium and that may introduce a significant error into thecalculated PBG level. Because the NBT method uses two readings atdifferent times following the addition of NBT to the specimen, it isalso referred to as "the kinetic method." However, this method does noteliminate the adverse effects caused by elevated levels of uric acidand/or bilirubin.

It is known to add polyethylene glycol to serum to precipitate bothglycated and non-glycated globulins while leaving glycated andnon-glycated albumin and smaller molecules (e.g., uric acid andbilirubin) in solution, i.e., in the supernatant, and then to test thesolution for glycated albumin by the nitroblue tetrazolium colorimetricmethod. That separation (precipitation) method leaves the non-protein,low molecular weight interfering substances in solution with thealbumin, possibly to interfere with the colorimetric test if theirlevels are high enough. It is also known to use affinity chromatographyon serum to separate glycated serum proteins (principally glycatedalbumin and glycated globulins) from the other substances present in theserum. These other substances do not bind to the column and includenon-glycated proteins and the non-protein, low molecular weightinterfering substances. The glycated proteins bound in the column areeluted and the protein concentration is measured by colorimetric ornon-colorimetric methods. Mashiba et al., "Measurement of GlycatedAlbumin by the Nitroblue Tetrazolium Colorimetric Method," ClinicaChimica Acta, vol. 212, pp. 3-15 (1992). See also Armbruster, Clin.Chem., vol. 33, no. 12, pp. 2153-2163 (1987). It is also known to adduricase to serum (to eliminate uric acid) along with the colorimetricreagent and then colorimetrically measure the glycated proteins in theserum.

Researchers disagree as to the effects of albumin on the NBT (nitrobluetetrazolium) test. Johnson et al., Clinica Chimica Acta, vol. 127, pp.87-95 (1982), indicate that in the NBT test, correcting fructosamine foralbumin concentration makes the difference between normal and diabeticsera less clear. On the other hand, Armbruster, Clin. Chem., vol. 33,no. 12, pp. 2153-2163 (1987), a survey article, reports at pp. 2157-2158conflicting opinions from other researchers. Some researchers said thathypoalbuminemia (clinically significant low albumin level) influencedthe NBT assay only when the albumin concentration was less than 30-35grams/liter. Other researchers found that protein-bound glucose valuesfrom the NBT method were affected by albumin regardless of the albuminconcentration and they suggested a certain correction factor based onalbumin concentration. Other researchers recommended a differentcorrection based on albumin concentration. Yet other researchers did notfind fructosamine to be significantly influenced by albuminconcentration.

Rosenthal et al., Clin. Chem., vol. 34, no. 2, pp. 360-363 (1988),report on a single-color-reading method for determining protein-boundglucose. At p. 362 they surmise that fructosamine concentrations fromtheir method are affected by serum albumin concentrations in the samemanner as the two-color-reading or kinetic method and that this shouldbe taken into account for patients whose serum albumin concentrationsare abnormal. Finally, Armbruster, Clin. Lab. Sci., vol. 3, no. 3, pp.184-188 (May/June 1990), another survey article, reports at p. 187 thatthe drawbacks of the NBT method include the nonspecific nature of thereaction in serum, the effects of albumin concentration, and how to bestcalibrate the test. Armbruster recommends that determining glycatedhemoglobin instead of determining protein-bound glucose may be moreadvantageous when a patient's albumin or total protein values are low(Id.).

Thus, there are conflicting opinions in the literature as to whetherdetecting protein-bound glucose in serum is a reliable and accurate wayto test for diabetes or to monitor the course of therapy, whether thepresence of albumin significantly affects the NBT test for protein-boundglucose and if so, how to compensate for that effect, and whether anassay for determining protein-bound glucose should be used at all forpatients who have low albumin levels.

Furthermore, no reliable method exists for eliminating or significantlyreducing the adverse effects of, e.g., elevated uric acid and/orbilirubin levels in PBG assays, particularly colorimetric assays.Accordingly, there is a continuing need for a reliable, reproducible,relatively rapid, and relatively inexpensive method for testingindividuals for diabetes with high accuracy and for monitoring thecourse of diabetes therapy, even if those individuals have levels ofuric acid, bilirubin, and/or other substances that would otherwiseinterfere with determination of their protein-bound glucose.

SUMMARY OF THE INVENTION

A method of testing individuals for diabetes that satisfies thoserequirements has now been developed. Other features and advantages ofthe method of this invention will be apparent to those skilled in theart from this disclosure.

One aspect of the invention concerns a method of reducing interferencefrom interfering substances in an assay for the presence ofprotein-bound glucose, said method comprising treating a compositioncontaining interfering substances and protein-bound glucose comprisingglycated albumin and other glycated proteins to produce a product to beused in the assay that as compared to the composition is substantiallyricher in glycated albumin and substantially poorer in interferingsubstances.

Another aspect of the invention concerns a method of determiningprotein-bound glucose that is principally glycated albumin, said methodcomprising:

(a) treating a composition containing protein-bound glucose andinterfering substances comprising uric acid, bilirubin, glutathione, andascorbic acid and its metabolites to produce a product that issubstantially free of those interfering substances; and

(b) colorimetrically assaying the product for protein-bound glucose.

Another aspect of the invention concerns a method of reducinginterference from interfering substances in an assay for the presence ofprotein-bound glucose, said method comprising contacting a compositioncontaining protein-bound glucose and one or more interfering substanceswith a reagent to produce a precipitate to be used in the assay thatcomprises protein-bound glucose that can be redissolved and that issubstantially free of one or more of the interfering substances.

Another aspect of the invention concerns a method of assessing thelikelihood of an individual in a population having diabetes even ifspecimens from the individual used for such assessment containinterfering substances that would otherwise tend to interfere with suchassessment, said method including the steps:

(a) obtaining from the individual a specimen that contains protein-boundglucose that is principally glycated albumin and which may contain oneor more interfering substances that may interfere with a subsequentassay for protein-bound glucose;

(b) treating the specimen to produce a product that as compared to thespecimen is substantially richer in protein-bound glucose andsubstantially poorer in one or more of the interfering substances;

(c) treating the product from step (b) to determine the level ofprotein-bound glucose for the individual;

(d) determining the glucose level for that individual; and

(e) comparing the levels of protein-bound glucose and glucose for theindividual to pre-established values that together tend to indicatewhether an individual is likely to have diabetes.

Another aspect of the invention concerns a method of assessing thelikelihood of an individual in a population having diabetes even ifspecimens from the individual used for such assessment containinterfering substances that would otherwise tend to interfere with suchassessment and in which the population has been divided into differentcategories based on their glucose levels and protein-bound glucoselevels, some of which categories are associated with a greater or alesser likelihood of having diabetes, said method including the steps:

(a) obtaining from the individual a specimen that contains protein-boundglucose that is principally glycated albumin and which may contain oneor more interfering substances that may interfere with a subsequentassay for protein-bound glucose;

(b) treating the specimen to produce a product that as compared to thespecimen is substantially richer in glycated albumin and substantiallypoorer in one or more of the interfering substances;

(c) treating the product from step (b) to determine the level ofprotein-bound glucose for the individual;

(d) determining the glucose level for that individual; and

(e) comparing the levels of protein-bound glucose and glucose for theindividual to pre-established values that together place the individualinto one of the categories.

In a preferred aspect of the invention, the specimen from the individualwhose diabetic status is being determined is treated, preferably with aprecipitating agent (e.g., uranyl acetate) to precipitate the glycatedalbumin, which typically accounts for about 80% of the protein-boundglucose in the specimen. The supernatant will contain substantially allof the interfering substances that were present in the specimen, and theprecipitate, which is substantially free of the interfering substances,will then be assayed for protein-bound glucose. "Substantially free ofinterfering substances" means that the level of interfering substancesin a material to be assayed is low enough so that those substances donot cause any clinically significant error in the assay forprotein-bound glucose. The clinical variables used herein (glucose,protein-bound glucose, and optionally albumin) are preferably determinedfrom blood serum (referred to herein as "serum"). Under appropriateconditions other blood derivatives may also be tested to determine oneor more of those three values. In another preferred aspect of theinvention, the protein-bound glucose is determined by a colorimetricmethod, preferably using an electron acceptor, which most preferably isa viologen such as benzyl viologen or methyl viologen.

Applicants believe that the evidence is not conclusive yet regardingwhether normalization is necessary or desirable if the individual inquestion has hypoalbuminemia. Thus, the method of this invention allowsthe value for protein-bound glucose to be used as such or to benormalized, e.g., to account for individuals having hypoalbuminemia. Onemethod of determining normalized protein-bound glucose for an individualis to determine the protein-bound glucose level, determine the albuminlevel, and divide the protein-bound glucose level by the albumin levelto yield the individual's normalized protein-bound glucose level("normalization"). The method of assessing the risk of having diabeteswill then use the individual's glucose value and either his or herprotein-bound glucose value or his or her normalized protein-boundglucose value.

The method of this invention can be used to preliminarily screen peoplefor diabetes or it can be used as a secondary test. Advantages of theinvention include the increased sensitivity and accuracy of the method,e.g., for detecting individuals who have diabetes but who have unusuallylow albumin levels and, thus, might be reported as normal in a standardNBT test for protein-bound glucose; for discriminating betweennon-fasting subjects who have and who do not have diabetes, all of whommight be reported as probable diabetics from the standard glucose test;for detecting specimens that may have been mishandled; and for moreaccurately determining the PBG level for an individual even if thatindividual (and thus a specimen taken from that individual) hassufficiently high levels of interfering substances. For example,elevated uric acid and/or bilirubin may cause erroneously high PBG,which in turn increases the likelihood that the patient will beerroneously reported to be diabetic. Increased accuracy in detectingwhether a patient has diabetes obviously reduces the number of follow-uptests (and associated cost and time) that would otherwise be necessaryand allows any necessary therapy to begin sooner.

The method of this invention for reducing the effect of interferingsubstances finds particular use when protein-bound glucose is to bedetermined by a colorimetric assay. In a colorimetric assay used in thisinvention, interfering substances are non-protein, low-molecular weightsubstances that could interfere with the colorimetric determination ofPBG and that are typically endogenous (but could also be exogenous) andare typically reducing substances, for example, uric acid, bilirubin,glutathione, ascorbic acid and its metabolites, and certain drugs (e.g.,quinones). Thus, preferably, the non-protein, low molecular weightsubstances that are substantially eliminated by the method of thisinvention from the composition or specimen to be further tested areselected from the group consisting of uric acid, bilirubin, glutathione,and ascorbic acid and its metabolites. Also preferably, the method ofthis invention removes a substantial portion of each of those substances(uric acid, bilirubin, glutathione, and ascorbic acid and itsmetabolites) from the composition or specimen to be further tested forglycated protein, i.e., the composition or specimen is substantiallyfree of each of those substances. By "substantially free" is meant thatat least 70%, desirably at least 80%, preferably at least 90%, morepreferably at least 95%, and most preferably at least 98% of theinterfering substances that are in the composition or specimen to betreated are not found in the product or precipitate after the treatment.Thus, most preferably, at least 70%, desirably at least 80%, preferablyat least 90%, more preferably at least 95%, and most preferably at least98% of each of the interfering substances selected from the groupconsisting of uric acid, bilirubin, glutathione, and ascorbic acid andits metabolites that are in the composition or specimen to be treatedare not found in the product or precipitate after the treatment.

Typically the glycated albumin is at least 50%, desirably at least 60%,preferably at least 70%, and more preferably about 80% of theprotein-bound glucose. "Product to be used in the assay" and "assayingthe product" refer to the fact that the product is used in the assay forprotein-bound glucose as is or after further treatment. For example, iftreating the composition results in a "product" that is a precipitate,it may be necessary to redissolve the precipitate (after it has beenseparated from the supernatant, which contains interfering substances)so that the subsequent assay for the protein-bound glucose, e.g., acolorimetric assay, can be performed (because colorimetric assaystypically are performed on liquids and not solids). "Precipitate to beused in the assay" refers to the fact that the precipitate is used as isor after further treatment.

Usually at least 70%, desirably at least 80%, preferably at least 90%,more preferably at least 95%, and most preferably at least 98% of theglycated albumin that is in the composition or specimen to be treated isfound in the product or precipitate after the treatment. Usually atleast 70%, desirably at least 80%, preferably at least 90%, morepreferably at least 95%, and most preferably at least 98% of theinterfering substances that are in the composition or specimen to betreated are not found in the product or precipitate after the treatment.Use of uranyl acetate as the precipitating agent usually results inobtaining the higher values for each of those ranges, i.e., with uranylacetate the amount of glycated albumin in the composition or specimenthat is found in the product or precipitate after treatment is usuallycloser to the 98% value than to the 70% value, and the amount ofinterfering substances not found in the product or precipitate isusually closer to the 98% value than to the 70% value.

BRIEF DESCRIPTION OF THE DRAWING

To facilitate further description of the invention, the accompanyingfigure is provided. The ordinate shows PBG level (in milligrams ofprotein-bound glucose in serum per deciliter of serum) for individualsin a population and the abscissa shows their respective glucose level(in milligrams of glucose per deciliter of serum). Thus, the point onthis graph for an individual will be determined by the individual's PBGand glucose values. The plot has been divided into six regions orcategories, indicated by I, II, III, IV, V, and VI. The significance ofeach category is described below. It should be understood that thedrawing is provided for descriptive purposes and should not be construedto unduly limit the scope of the claims.

DETAILED DESCRIPTION OF THE INVENTION

Any clinically acceptable assay for glucose and protein-bound glucose(and albumin, if determined and used to normalize the value forprotein-bound glucose) may be used. However, the method of thisinvention for reducing the effect of interfering substances findsparticular use when protein-bound glucose is to be determined by acolorimetric assay. In a colorimetric assay used in this invention,interfering substances are typically endogenous low-molecular weightreducing substances that could interfere with the colorimetricdetermination of PBG, for example, uric acid, bilirubin, glutathione,ascorbic acid and its metabolites, and certain drugs (e.g., quinones).

Desirably, the blood derivative on which the assays are performed isserum. Assays for determining glucose, protein-bound glucose, andalbumin in the serum of an individual are well-known. When using thepreferred method of reducing the effect of interfering substances in thePBG assay, that is, to use uranyl acetate as a precipitating agent toprecipitate PBG and leave one or more of the interfering substances inthe supernatant, the specimen for the PBG assay will preferably be serumthat is not hemolyzed or lipemic. Desirably, a colorimetric test is usedfor determining protein-bound glucose ("PBG"), which test may beautomated and run at the same time the glucose and albumin tests arerun. Desirably, all tests are automated and all are run withsubstantially the same equipment, desirably a multi-channel instrument.A single specimen may be used and subdivided for the various tests.After uranyl acetate or other precipitating agent has been added to aspecimen, glucose and albumin assays desirably are not run on thatspecimen or on the resulting supernatant.

The protein-bound glucose test used to determine PBG desirably is anautomated colorimetric in vitro assay for quantifying the amount ofglucose covalently linked to the lysine and terminal amino groups ofvarious serum protein, the principal one of which is albumin. Theattachment of glucose to the amino groups of proteins is anon-enzymatic, post-translational process often referred to in moderntechnical literature as "protein glycation."

As explained above, circulating plasma proteins may becomenon-enzymatically glycated in the same manner as the hemoglobin of redblood cells. Glycation is a slow, continuous, and irreversible reactionwhose rate and extent depend principally on the glucose concentration towhich the proteins are exposed. When serum proteins are glycated invivo, glycated albumin usually accounts for about 80 percent of theglycated serum proteins (that is, protein-bound glucose). See, e.g.,Dolhofer et al., Clinica Chimica Acta, vol. 112, pp. 197-204 (1981);U.S. Pat. No. 4,956,301, column 10, line 8 et seq. Just as withglycohemoglobin (glycated hemoglobin), the amount of protein-boundglucose increases in patients with poorly controlled diabetes.

A number of studies have documented that protein-bound glucose levelscan be elevated despite seemingly normal levels of glycohemoglobin. Thatis consistent with the fact that the half-life of albumin and,therefore, of glycated albumin is shorter, about 19 days, whereas thehalf-life of hemoglobin and, therefore, of glycated hemoglobin is about60 days. Accordingly, a patient's glycated hemoglobin level, whichindicates long-term glycemia, may be normal whereas the protein-boundglucose level, which indicates intermediate-term glycemia, may beelevated. Thus, determining protein-bound glucose may provide an earlywarning of the onset of diabetes or an indication of transienthyperglycemia, which may not be revealed by subsequent glycohemoglobinassay.

In the method of this invention, the normalized PBG level of theindividual may be used, although normalization may not be necessary,even for individuals who have hypoalbuminemia. Any method ofnormalization can be used provided it allows for and facilitatesdetecting the existence of diabetes in a patient using the method ofthis invention even if the patient also has hypoalbuminemia.Normalization is preferably accomplished by dividing the assay value forPBG level by the assay value for albumin level. Another method ofnormalizing is dividing the raw PBG level by a factor proportional tothe albumin level, for example, dividing the PBG level by half of thealbumin level. Another method is to categorize individuals based ontheir albumin levels, assign a different factor to each albumin level,and multiply or divide the raw PBG level for an individual by the factorfor that individual corresponding to that individual's albumin level.

The chemistry of glycation and of the preferred PBG assay are furtherdescribed as follows. In the first step of glycation, the carbonyl groupof glucose combines with the amino groups of proteins to form aldimine(1-amino-1-deoxyglucose) intermediates known as Schiff bases. In thesecond step, the labile aldimine-Schiff base intermediates undergo theAmadori rearrangement (the isomerization of aldosylamine to a1-amino-1-deoxyketose) to yield a relatively stable ketoamine adduct tothe protein. The straight-chain ketoamine adduct is thought to undergocyclization to a more stable hemiketal furanose or pyranose ringstructure.

Ketoamine-linked N-(1-deoxyfructos-1-yl) groups, the Amadorirearrangement products when the Schiff base originates from glucose, areelectron donors (reducing substance) at alkaline pH levels. The reducingpower of these Amadori rearrangement products in dilute NaOH at roomtemperature has been demonstrated by the conversion of o-dinitrobenzeneto a purple color after one minute; by rapidly decolorizing methyleneblue and dichlorophenolindophenol; and by reduction of Fehling solution,Tollen's reagent, nitroblue tetrazolium, and other tetrazolium salts.

The preferred PBG assay is based on the ability of Amadori rearrangementproducts to transfer electrons under alkaline conditions, in thepresence or absence of oxygen, to a colorimetric reagent, which is amixture of colorless, low-potential redox indicators widely used asartificial electron carriers in enzymic systems. The electron acceptorreagent preferably used herein comprises 1,1'-dibenzyl-4,4'-bipyridiniumdichloride (benzyl viologen); 1,1'-dimethyl-4,4'-bipyridinium dichloride(methyl viologen); and heterocyclic azole compounds as chromaphoricstabilizers. The transfer of electrons to the reagent results in theformation of blue, stable, free-radical cations (wavelength of maximumabsorbance is 540 nanometers). The absorbancy of the solution containingthe blue-colored reduced compounds is proportional to the amount ofprotein-bound glucose in the patient's sample.

The reagent typically will be stored as a dry powder. When reconstitutedwith deionized water, the aqueous reagent solution preferably used inthis assay will contain 212 milligrams per liter of the above-describedelectron acceptor reagent in a molar ratio of 1 part of benzyl viologen,1 part of methyl viologen, and 0.1 part of chromaphoric stabilizer. Atetrazolium compound may be used as the stabilizer and preferablytriphenyl tetrazolium chloride is used.

As used in the preferred PBG assay, the aqueous solution of electronacceptor reagent also contains 10.4 grams/liter of sodium carbonate and5.5 grams/liter of sodium bicarbonate. With these quantities, the pH ofthe reagent solution is 10.5 and the pH of the reaction mixture(specimen plus reagent solution) is 10.5. The pH of the solution may beincreased or decreased by increasing or decreasing the quantities ofcarbonate and bicarbonate used. It has been found that the pH of thereaction mixture should be at least about 10.0 so that the reduction ofthe electron acceptor compounds (the viologens) will proceedsufficiently rapidly. However, if the pH is too high, other compounds(for example, glucose) in the specimen will also react with the electronacceptor compounds to produce a greater intensity of color than isotherwise due to the PBG. This interference becomes noticeable whenusing NBT as the coloring agent if the pH is higher than about 10.8.With the preferred electron acceptor reagent described above, suchinterference by glucose, for example, is not significant until the pH isgreater than about 12.6. Accordingly, because any acceptable coloringagent may be used in a colorimetric method for detecting PBG, oneskilled in the art will know that the pH of the aqueous coloring agentshould be adjusted so that it is sufficiently alkaline for the reactionto proceed satisfactorily but not so alkaline that interference byglucose or other compounds present in the specimen that have not beeneliminated by the previous separation (e.g., precipitation) becomessignificant.

The aqueous reagent solution described above should be stored in a darkbottle and held at 2°-8° C. Under those conditions, the aqueous reagentsolution will be stable for at least five days and possibly for up to 30days. The preferred dry reagent described above is available fordetermining PBG from National Screening Institute of Metuchen, N.J.

The preferred PBG assay used in the method of this invention is run at37° C. and preferably is carried out automatically on a serum specimenusing either a Hitachi 736-50 Analyzer or an Olympus AU5000 Analyzer.The following operating parameters have been found to be satisfactoryfor each of the two analyzers:

    ______________________________________                                        Hitachi 736-50 Analyzer Instrument Parameters                                 Channel No.:       # PBG                                                      Assay Code:        ENDPOINT-20-20                                             Sample Volume:     15                                                         R1 Volume:         250-9999                                                   R2 Volume:         0-9999                                                     Wavelength 1:      700 NM                                                     Wavelength 2:      546 NM                                                     Reagent Blank Absorption:                                                                        -1-0                                                       Reagent Blank Concentration:                                                                     0                                                          Standard Concentration:                                                                          Use Assayed Value Of                                                          Bovine Serum Calibrator                                    Recalibrate (Blank):                                                                             1                                                          Recalibrate (Standard):                                                                          3                                                          Factor:            4267                                                       Unit Factor:       1.00                                                       Standard Absorption:                                                                             10%                                                        Allowance                                                                     Normal Range Low:  0                                                          Normal Range High: 9999                                                       Absorption Limit (Rate):                                                                         0 (Increase)                                               Olympus AU5000 Analyzer Instrument Parameters                                 Operation:         Yes                                                        Sample Volume:     15 microliters                                             Reagent Volume                                                                R1:                250 microliters                                            R2:                0                                                          W3 Operation                                                                  R1:                Yes                                                        R2:                No                                                         Method:            End                                                        Wavelength 1:      540                                                        Wavelength 2:      750                                                        Reaction Slope:    +                                                          Measuring Point                                                               Start:             8                                                          End:               8                                                          OD Value Range                                                                Max.:              N/A                                                        Min.:              N/A                                                        Limit Of Linearity:                                                                              N/A                                                        Repeat Range                                                                  High:                                                                         Low:                                                                          QC Group                                                                      1:                 1                                                          2:                 2                                                          Reagent OD Range                                                              Max.:              2.000                                                      Min.:              -0.1000                                                    ______________________________________                                    

Usually only one intensity measurement is needed with the preferred PBGassay because desirably the interfering substances have beensubstantially removed from the product or precipitate on which the PBGassay is run. Alternatively, two, three, or more intensity readings maybe utilized. However, more than two measurements may unduly increase thetotal time required for running a PBG assay. Generally, the time fortaking a single reading will be 5-20 minutes, desirably 5-15 minutes,and preferably eight minutes after the reagent solution and treatedspecimen (desirably the redissolved precipitate from the precipitationstep) are combined to form the reaction mixture. When using automatedequipment such as the above-described Hitachi and Olympus analyzers,eight minutes is the preferred time for a single reading or for the lastreading when two or more readings are used. That is because eightminutes is the maximum set time for those machines. The optimum timesfor taking the reading(s) for a particular PBG colorimetric assay withparticular equipment can readily be determined. As will be understood byone skilled in the art, the times will be affected by which colorimetricreagent is used, its concentration in the reaction mixture, the pH andtemperature of the reaction mixture, and the equipment employed.

For quality control of the PBG assay, control sera from NationalScreening Institute are preferred. MAS Control I and MAS Control III,available from Medical Analysis Systems Inc. of Camarillo, Calif., mayalso be used. Any other satisfactory control sera may be used. The MAScontrols are liquid human sera and should be stored according to themanufacturer's recommendations. In one study, MAS Control I produced amean value of 3.91 milligrams of PBG per deciliter of serum (with arange of 3.76-4.12 mg/dl) and MAS Control III yielded a mean value of6.55 milligrams PBG/deciliter of serum (with a range of 6.38-6.72mg/dl).

Use of commercially available control sera may result in unusually highPBG values, for the following reason. In making control sera,manufacturers usually pool large amounts of sera and allow the sera tosit for extended periods of time. During these lengthy periods, theglucose in the sera continuously reacts with the serum proteins. Thus,these control sera may have abnormally high PBG levels. Although it isdifficult and expensive to prepare serum controls that have not sufferedsuch glycation, the control sera from National Screening Institute yieldnormal PBG values. Such sera are produced by collecting fresh serum fromnon-diabetics, dialyzing the serum to remove endogenous glucose, andlyopholizing the serum. Obviously, control sera that give normal PBGvalues are preferred and can be prepared by anyone skilled in the art.

It has also been found that hemolysis interferes with the preferred PBGassay and, therefore, the preferred method of this invention should notbe performed using hemolyzed serum or with lipemic serum.

With respect to calibration standards for the preferred PBG assay,samples of bovine calibrator are available from National ScreeningInstitute, which assigns a calibration value (milligrams of PBG perdeciliter of reconstituted calibrator). National Screening Instituteobtains the calibrator from the Gilford division of Ciba-CorningDiagnostics, Oberlin, Ohio. Gilford Calibrator Catalog No. 606-871-2 hasbeen found to be satisfactory. The HPLC method of Schleicher et al., J.Clin. Chem. Clin. Biochem., vol. 19, pp. 81-87 (1981), has been found tobe satisfactory for assigning a calibration value to the Gilford bovinecalibrator. Other suitable calibration standards and methods may beused. It has been found that the Gilford calibrator should be storedunder refrigeration (for example, at 2°-8° C.) until its expirationdate. The calibrator may be reconstituted with the ionized water andmixed gently, for example, for 30 minutes. The reconstituted calibratorif stored properly (for example, at 2°-4° C.) is stable for 24 hours.

In a study of about 2,600 patients using the Hitachi 736-50 and OlympusAU5000 analyzers, the normalized PBG reference range was found to beless than 1.20 milligrams PBG per gram of albumin. Thus, for the patientpopulation surveyed the cut-point for the upper limit of normal for theratio of protein-bound glucose to albumin (that is, normalizedprotein-bound glucose) was found to be 1.2 milligrams PBG/grams albumin.In interpreting the results of the preferred PBG assay, specimens whosenormalized PBG levels are less than 0.3 mg PBG/g albumin or greater than4.0 mg PBG/g albumin should be retested. That study was performed onspecimens that were not subjected to the separation step to eliminateinterfering substances prior to the PBG colorimetric assay.

It has been found for the preferred colorimetric PBG assay using thepreferred colorimetric reagent that uric acid levels equal to or above8.5 milligrams/deciliter cause the PBG values determined by that assayto be higher than the true value by 10% or more and that bilirubinlevels equal to or above 2.0 milligrams/deciliter also cause a 10% orgreater error. Despite the general practice of not using colorimetricmethods to determine PBG if the patient has elevated uric acid and/orbilirubin levels, it has been found that PBG level can successfully beused as one of the two key indicators in the method of this invention(in addition to the glucose level) provided it is determined from aspecimen that has been treated using the separation step of thisinvention. The thus-obtained PBG value and the glucose value (the otherkey indicator) are utilized together to place the individual into one ofseveral categories that have been previously established for thepopulation and against which the individual is to be compared.

Using the combination of serum glucose and serum PBG (optionallynormalized) obtained in accordance with this invention is superior tousing glucose alone or PBG (or normalized PBG) alone in testing fordiabetes. The combination allows a physician to determine whether aserum glucose result is representative of glycemia over the previous 1-2weeks, better interpret the glycemic status of non-fasting subjects(because PBG is an integrated measure of the average level of glycemiaover the previous two weeks or so), and determine that significantperiods of hyperglycemia have been present in diabetic patients who maypresent normal serum glucose. Eliminating interfering substances priorto assaying for PBG allows a more accurate PBG value to be obtained andused in conjunction with glucose level, with concomitant increase in theaccuracy of diagnosis or assessment. Other advantages of using thecombination will be apparent from further consideration of the figure.

The PBG/glucose plot of the accompanying figure is divided into sixsections or categories. The dark vertical line separating sections I andIII from sections V and VI is drawn at the cut-point for the populationsurveyed for the lower limit of normal for glucose level, which in thiscase is established at about 65 milligrams of glucose per deciliter ofserum. The dark vertical line separating sections I and III fromsections II and IV is drawn at the cut-point for the population for theupper limit of normal for glucose level, which in this case isestablished at about 130 milligrams of glucose per deciliter of serum.The dark horizontal line separating sections I, IV, and V from sectionsII, III, and VI is drawn at the cut-point for the population for theupper limit of normal for the ratio of protein-bound glucose to albumin(normalized PBG), which in this case is established at about 1.2milligrams of PBG per gram of albumin.

Obviously the values at which the cut-points are established will vary,depending on a number of factors, including which assays are used, thecalibration and standardization values, the equipment used, the units inwhich the glucose and normalized PBG levels are expressed, and, if used,the method employed to normalize the PBG level for albumin level. Thecut-points may also vary depending upon the genetic background of thepopulation surveyed and their diets. Other factors may also affect thecut-points established, such as what a clinician considers "normal" forglucose level and PBG level. Establishing a cut-point for the upperlimit of normal for the PBG value is well within the skill of the art asare establishing the cut-points for the upper and lower limits of normalfor glucose level.

After the figure or equivalent (e.g., computer database) is prepared forthe reference population, it can then be used as the basis for making anassessment of the likelihood that a patient has diabetes. A patientwhose glucose value and PBG value place him or her within category Iprobably does not have diabetes (or if he or she is a known diabetic,the course of therapy is being successful) because the glucose and PBGlevels are consistent with normoglycemia (normal glycemia). A patientwhose glucose and PBG level place him or her in region II of the figurehas a higher than average probability of having diabetes because his orher glucose and PBG levels are consistent with clinically significanthyperglycemia. The patient should be retested with a standardconfirmatory test.

A patient whose glucose and PBG levels place him or her within sectionIII of the figure has normal glucose but elevated PBG and may bediabetic. A glucose test alone would not reveal the possible diabetes.

A patient whose glucose and PBG levels place him or her in category IVhave elevated glucose levels but normal PBG levels. That indicatestransient hyperglycemia. Such a patient was probably normoglycemic overthe past two weeks. The elevated glucose level may indicate thecontemporaneous onset of diabetes or that the patient did not fast for asufficiently long time before his or her blood specimen was taken orthat he or she has recently been on low-carbohydrate diets. For suchpatients the standard glucose test alone would indicate the need forfurther testing, which might unduly worry them. However, because bothglucose and PBG levels are determined and because the PBG levels are notabove the established cut-point, a physician may advise patients whoappear to belong in category IV that they probably do not have diabetesand/or make further inquiry concerning their diet and whether theyfasted before the blood sample was taken.

A patient placed in category V exhibits low glucose level and normal PBGlevel. This suggests hypoglycemia or possible specimen mishandling.Specimens from such patients may have individuals may have suffered invitro glycolysis because, as discussed above, the glycation reactionbetween blood proteins and glucose proceeds continuously. If a specimenis inadvertently allowed to stand too long before being assayed, theglucose level will fall.

A patient placed in category VI exhibits low glucose level and elevatedPBG level. That suggests possible hypoglycemia or possible mishandlingof the specimen. As explained, if sufficient time elapses before aspecimen is analyzed, the glucose level may fall significantly and thePBG level may rise significantly as glycation occurs. If such a delay inprocessing the specimen has not occurred, the results suggest that thepatient may have had significant hyperglycemia during the previous twoweeks. A patient assigned to category V or VI probably should beretested within a week or two.

Patients who have sufficiently elevated uric acid and/or bilirubin toresult in erroneously high PBG values if the separation step of thisinvention were not employed might as a result be placed in category II;however, the method of this invention tends to eliminate suchinterfering substances prior to the PBG assay and thereby tends toresult in those patients being correctly placed instead in category IV,thereby resulting in a change of diagnosis.

It is not necessary that the reference population be divided into sixcategories; a greater or lesser number of categories may be useddepending on how many cut-points are established. For example, acut-point for the lower limit of normal for the glucose level need notbe established or utilized.

In the preferred separation of this invention for reducing the effect ofinterfering substances on the subsequent PBG assay, preferably aprecipitating agent is used, most preferably uranyl acetate. Thus, thespecimen, preferably serum, is combined with the precipitating agent.The resulting precipitate is separated from the supernatant, theprecipitate is redissolved, and the assay (preferably colorimetric), isrun on the resulting solution.

The precipitate will contain glycated protein (PBG) that issubstantially glycated albumin. The supernatant will containsubstantially all of the interfering substances. It is important thatthe precipitating agent not denature the protein of the precipitate(principally albumin) otherwise it will be difficult or impossible toredissolve the precipitate to obtain a solution of glycated protein onwhich to run the PBG assay.

Uranyl acetate is the preferred precipitating agent. It breaks ionicbonds between albumin and other substances (e.g., the interferingsubstances), does not break covalent bonds between albumin and othersubstances (e.g., the sugar-albumin bonds of the glycated albumin), andprecipitates the glycated albumin without denaturing the albumin,thereby permitting dissolution of the precipitate for the PBG assay,which is desirably a colorimetric assay.

The preferred precipitating agent is preferably used as an aqueoussolution in which 600 microliters of solution contain 4.1 millimoles/1of uranyl acetate. The solution is buffered to a pH of 4.1 using sodiumacetate buffer and it contains 1% by volume of methanol as a stabilizer.

Albumin accounts for about 80 percent of the glycated proteins in theserum of almost all people. Thus, separating glycated albumin from theother glycated proteins in the specimen (e.g., glycated globulins) doesnot affect the validity of using the PBG value obtained on materialresulting from the separation step of this invention instead of using aPBG value that would otherwise be obtained on the specimen without anyseparation step. Furthermore, as explained above, without the separationstep of this invention, interfering substances would tend to result inerroneous PBG values.

The use of the method of this invention is illustrated as follows.Forty-five microliters each of calibrator, controls I and II, andpatient samples are pipetted in polypropylene microcentrifuge tubes. Sixhundred microliters of the preferred precipitating agent solution areadded to each tube, the tubes are capped, and the contents are mixed bya vortex mixer. The capped tubes are placed in a microcentrifuge (e.g.,HERMLE model BHG) and are centrifuged at 15,000 rpm for 3 minutes atroom temperature. The contents are decanted and the supernatant isdiscarded by inverting the tubes and tapping the open ends a few timesto remove any trace of supernatant. To the remaining precipitate in eachtube is added 1.5 milliliters of the preferred colorimetric indicatordescribed above. The tubes are capped and placed in a heating block heldat 37° C. After 15 minutes, the tubes are mixed one at a time by vortexmixing after which mixing each tube is immediately returned to theheating block. It is important that the precipitate in each tube becompletely redissolved. At the end of 30 minutes total for each tube,each tube is removed from the heating block and the contents are mixedby inverting the tube three times. Using a spectrophotometer that hasbeen "zeroed" against water, the absorbance of the contents of each ofthe tubes is measured at 538 nanometers after the contents has beentransferred to an appropriate cuvette.

The data are then manipulated as illustrated by the following todetermine the PBG values for the unknowns. Assume that the absorbance ofthe calibrator has been determined to be 0.181, that of control I(non-diabetic) to be 0.155, and that of control II (diabetic) to be0.327. The PBG value for control II is thus 0.327/0.181, which equals1.81. The PBG value for patient 1, who had an absorbance reading of0.186 is 0.186/0.181, which equals 1.03, and the PBG value for patient2, who had an absorbance reading of 0.288 is 0.288/0.181, which equals1.59. The PBG values for patients 1 and 2 in combination with theirindividual glucose values will allow an assessment to be made as to thelikelihood of each of them having diabetes, for example, by placing eachof them into one of the six categories shown in the figure.

PBG values for 191 patients obtained using the method of this invention(with the separation step) were compared to their Hemoglobin A1c valuesobtained using the commercially available Biorad Diamat System Analyzer.The overall coefficient of correlation (r) between the thus-determinedPBG and Hemoglobin A1c values for the 191 patients was found to be 0.91,which is excellent correlation.

As will be obvious to one skilled in the art, use of PBG obtained usingthe separation method of this invention and glucose together tocharacterize the patient's biochemistry for assessment significantlyenhances a physician's picture of the patient regarding possiblediabetes and allows the physician to provide additional information tothe patient. For example, for patients whose glucose levels and PBGlevels place them in category IV, a physician can ask them to beretested without alarming them by telling them that they probably do nothave diabetes but that for some reason the glucose levels were unusuallyhigh. Without knowing he or she displayed a normal PBG value, anindividual who had a glucose level over 200 mg/dl might be undulyalarmed if asked to submit to further testing. Similarly, an individualwhose glucose level appeared to be normal, for example, slightly over100 mg/dl, would not be detected as probably having diabetes andtherefore needing further testing if his or her PBG value of just over2.0 were not also provided to the physician. If that individual alsosuffered from hypoalbuminemia, the PBG value might or might not placethe individual under the cut-point of the upper limit of normal for PBGvalues (i.e., in category I) whereas normalization might obviate thatproblem.

Variations and modifications in this invention will be apparent to thoseskilled in the art and the claims are intended to cover all suchvariations and modifications that fall within the true spirit and scopeof the invention. For example, it is not necessary that graphicalmethods be used with the PBG (optionally normalized) and glucose levelsto divide the reference population into categories. Any method can beused for such categorization and the different categories or groupingsneed not even be called "categories" or the like. What is common aboutall of the methods that can be used is that combinations of the twovariables (glucose and PBG) for an individual being tested when comparedto the appropriate reference values for the reference population allowthat individual's probable risk of having diabetes to be assessed. Thus,for example, cut-points for the reference population can be establishedwithin the ranges of values for PBG level (optionally normalized) andglucose level, and the cut-points can be chosen so that values above orbelow the cut-points have some clinical significance to facilitate suchassessment.

It should be understood that these cut-points for "normal" may notstrictly correspond to what physicians would in the abstract considernormal. For example, a cut-point may be chosen to err on the side ofsafety so that those who would otherwise be classified as borderline butstill "normal" are identified as possibly needing further testing.

It should also be understood that the reference population cut-pointsfor any one of the variables may vary with the values for the othervariables, e.g., the upper limit of normal for glucose may vary with thePBG level. In graphical terms, with reference to the figure, that wouldmean, for example, that the straight vertical line separating regions Iand III from regions II and IV might instead be established as a curvedline, or a diagonal straight line, or a partially vertical/partiallycurved or diagonal line, or a combination thereof, and the same is truefor any of the other cut-points.

We claim:
 1. A method of determining protein-bound glucose in a specimencomposition containing protein-bound glucose and one or more interferingsubstances that may interfere with an assay for protein-bound glucose,said method comprising the steps:(a) contacting at a pH of about 4.1 thespecimen composition with a first reagent composition containing about4.1 millimoles/liter of uranyl acetate to precipitate protein-boundglucose without denaturing it to produce (i) a precipitate productcomprising protein-bound glucose that can be redissolved and that issubstantially free of the one or more interfering substances and (ii) asupernatant containing the one or more interfering substances; (b)separating the precipitate product from the supernatant; (c) dissolvingthe precipitate product to give a solution of redissolved protein-boundglucose that is substantially free of the one or more interferingsubstances; and (d) contacting the redissolved protein-bound glucose insolution at a pH of at least about 10.0 with a colorimetric reagent thatcolorimetrically indicates the presence of protein-bound glucose.
 2. Themethod of claim 1 wherein the one or more interfering substances areselected from the group consisting of uric acid, bilirubin, glutathione,ascorbic acid, and its metabolites.
 3. The method of claim 1 wherein theprotein-bound glucose is principally glycated albumin.
 4. The method ofclaim 1 wherein the colorimetric reagent comprises an electron acceptor.5. The method of claim 4 wherein the electron acceptor is selected fromthe group consisting of benzyl viologen and methyl viologen.
 6. Themethod of claim 4 wherein the colorimetric reagent comprises benzylviologen and methyl viologen.
 7. The method of claim 1 wherein step (a)is conducted at room temperature.
 8. The method of claim 1 wherein step(c) is carried out at about 37° C.
 9. The method of claim 1 whereinsteps (c) and (d) are performed simultaneously.
 10. A method ofdetermining protein-bound glucose in a specimen composition containingprotein-bound glucose and one or more interfering substances selectedfrom the group consisting of uric acid, bilirubin, glutathione, ascorbicacid, and its metabolites, said method comprising the steps:(a)contacting at a pH of about 4.1 the specimen composition with a firstreagent composition containing 4.1 millimoles/liter of uranyl acetate toprecipitate protein-bound glucose without denaturing it to produce (i) aprecipitate product comprising protein-bound glucose that can beredissolved and that is substantially free of the one or moreinterfering substances and (ii) a supernatant containing the one or moreinterfering substances; (b) separating the precipitate product from thesupernatant; and (c) dissolving the precipitate product to give asolution of redissolved protein-bound glucose that is substantially freeof the one or more interfering substances while contacting theredissolved protein-bound glucose in solution at a pH of at least about10.0 with a colorimetric reagent that colorimetrically indicates thepresence of protein-bound glucose.
 11. The method of claim 10 whereinthe colorimetric reagent comprises benzyl viologen and methyl viologen.12. A method of determining whether an individual has diabetes thatutilizes both the protein-bound glucose value and the glucose value forthe individual, even if a specimen composition from the individual usedfor determining the protein-bound glucose value contains one or moreinterfering substances that may interfere with the determination of theprotein-bound glucose value, said method comprising the steps:(a)obtaining from the individual a specimen composition that containsprotein-bound glucose and also contains one or more interferingsubstances that may interfere with a subsequent assay for protein-boundglucose; (b) contacting at a pH of about 4.1 the specimen compositionwith a first reagent composition containing about 4.1 millimoles/literof uranyl acetate to precipitate protein-bound glucose withoutdenaturing it to produce (i) a precipitate product comprisingprotein-bound glucose that can be redissolved and that is substantiallyfree of the one or more interfering substances and (ii) a supernatantcontaining the one or more interfering substances; (c) separating theprecipitate product from the supernatant; (d) dissolving the precipitateproduct to give a solution of redissolved protein-bound glucose that issubstantially free of the one or more interfering substances; (e)contacting the redissolved protein-bound glucose in solution at a pH ofat least about 10.0 with a colorimetric reagent that colorimetricallyindicates the presence of protein-bound glucose to determine theprotein-bound glucose value for that individual; (f) determining theglucose value for that individual; and (g) determining whether thatindividual has diabetes using both the protein-bound glucose value andthe glucose value for that individual.
 13. The method of claim 12wherein the one or more interfering substances are selected from thegroup consisting of uric acid, bilirubin, glutathione, ascorbic acid,and its metabolites.
 14. The method of claim 12 wherein theprotein-bound glucose is principally glycated albumin.
 15. The method ofclaim 12 wherein the colorimetric reagent comprises an electronacceptor.
 16. The method of claim 15 wherein the electron acceptor isselected from the group consisting of benzyl viologen and methylviologen.
 17. The method of claim 15 wherein the colorimetric reagentcomprises benzyl viologen and methyl viologen.
 18. The method of claim12 wherein step (b) is conducted at room temperature.
 19. The method ofclaim 12 wherein step (d) is carried out at about 37° C.
 20. The methodof claim 12 wherein steps (d) and (e) are performed simultaneously. 21.A method of determining whether an individual has diabetes that utilizesboth the protein-bound glucose value and the glucose value for theindividual, even if a specimen composition from the individual used fordetermining the protein-bound glucose value contains one or moreinterfering substances that may interfere with the determination of theprotein-bound glucose value, the interfering substances being selectedfrom the group consisting of uric acid, bilirubin, glutathione, ascorbicacid, and its metabolites, said method comprising the steps:(a)obtaining from the individual a specimen composition that containsprotein-bound glucose and also contains one or more interferingsubstances that may interfere with a subsequent assay for protein-boundglucose; (b) contacting at a pH of about 4.1 the specimen compositionwith a first reagent composition containing 4.1 millimoles/liter ofuranyl acetate to precipitate protein-bound glucose without denaturingit to produce (i) a precipitate product comprising protein-bound glucosethat can be redissolved and that is substantially free of the one ormore interfering substances and (ii) a supernatant containing the one ormore interfering substances; (c) separating the precipitate product fromthe supernatant; (d) dissolving the precipitate product to give asolution of redissolved protein-bound glucose that is substantially freeof the one or more interfering substances; (e) contacting theredissolved protein-bound glucose in solution at a pH of at least about10.0 with a colorimetric reagent that colorimetrically indicates thepresence of protein-bound glucose to determine the protein-bound glucosevalue for that individual, the colorimetric reagent comprising benzylviologen, or methyl viologen, or both; (f) determining the glucose valuefor that individual; and (g) determining whether that individual hasdiabetes using both the protein-bound glucose value and the glucosevalue for that individual.